Purified phosphodiesterase from bovine and ovine brain exhibited a single peptide of about 59,000 daltons on sodium dodecyl sulfate gel electrophoresis. Limited proteolysis with chymotrypsin produced peptides of 57,000 and 45,000 daltons (bovine) and 55,000, 53,000 and 38,000 daltons (ovine). The final stable peptides (45K bovine, 38K ovine) were fully active without calmodulin and calcium. Substrate, cGMP, or cAMP, with or without magnesium, had no effect on the pattern of proteolysis. Glycerol (20%) markedly reduced the rate of proteolysis but did not alter thepattern. In the presence of calmodulin plus calcium, however, the final stable peptides were 47,000 daltons (bovine) and 43,000 daltons (ovine). These results suggestdifferences in the structure of phosphodiesterases of bovine and ovine brain. They further provide direct demonstration of a conformational change in phosphodiesterase in the presence of calmodulin plus calcium with alters the peptide bonds that are accessible to chymotryptic cleavage. Effects of the phosphodiesterase inhibitor trifluoperazine on proteolytic cleavage have also been investigated.